Monomer Arrangement in HSP90 Dimer as Determined by Decoration with N and C-Terminal Region Specific Antibodies

Maruya, Mikako; Sameshima, Masazumi; Nemoto, Takayuki K.; Yahara, Ichiro

doi:10.1006/jmbi.1998.2349

Cited by 71 publications

(74 citation statements)

References 23 publications

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“…The advent of the C-terminal crystal structure provided further evidence for the antiparallel dimeric architecture of Hsp90 that had been previously predicted by electron microscopy [20][21][22]. C-terminal truncations of Hsp90 abolish its ability to hydrolyze ATP, indicating that its dimeric nature is essential for its activity [13].…”

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confidence: 68%

Hsp90—From signal transduction to cell transformation

Brown

Zhu

Schmidt

et al. 2007

Biochemical and Biophysical Research Communications

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The molecular chaperone, Hsp90, facilitates the maturation and/or activation of over 100 'client proteins' involved in signal transduction and transcriptional regulation. Largely an enigma among the families of heat shock proteins, Hsp90 is central to processes broadly ranging from cell cycle regulation to cellular transformation. Here we review the contemporary body of knowledge regarding the biochemical mechanisms of Hsp90 and update the most current paradigms defining its involvement in both normal and pathological cell physiology. KeywordsHsp90; heat shock protein; molecular chaperone; ATPase; genetic capacitor Hsp90 defines a family of molecular chaperones that are highly conserved from prokaryotes to eukaryotes [1][2][3][4][5]. Nonessential for normal growth in most bacteria, Hsp90 is abundantly expressed in higher eukaryotes where it has been shown to be necessary for viability [6,7]. It functions as a homodimer that associates with co-chaperones to catalyze the maturation and/ or activation of over 100 substrate proteins that are known to be involved in cell regulatory pathways [5]. These 'client proteins' include protein kinases, nuclear hormone receptors, transcription factors, and an array of other essential proteins [8]. While much is known regarding the ATPase-driven conformational cycling of Hsp90, the precise physical effects imparted by this chaperone that serve to activate its substrates are still poorly understood [5]. Hsp90 architectureThree highly conserved domains comprise the structure of Hsp90. These include the N-terminal domain, responsible for ATP-binding, a proteolytically resistant core domain, and the Cterminal domain that facilitates homodimerization (Fig. 1a) [9]. In eukaryotes, a more variable charged region links the N-terminal domain to the core domain. The length and composition of this linker region is highly divergent among organisms [10]. As no atomic resolution structure for full-length Hsp90 is yet available, the most thorough structural analyses for Hsp90, to date, have been based on crystallographic studies of its individual domains.A mechanistic understanding of Hsp90 was nebulous until partial sequence homology was recognized between its N-terminal domain and two types of ATP-dependent proteins. These

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confidence: 68%

Hsp90—From signal transduction to cell transformation

Brown

Zhu

Schmidt

et al. 2007

Biochemical and Biophysical Research Communications

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“…Recent studies (9,25,26,34,44) indicate that ATP binding by hsp90 induces dimer contacts near the amino terminus, and this may be a key conformational transition relating to substrate-chaperone interaction. Also, monomeric yeast hsp90 fragments lacking regions toward the carboxyl terminus exhibit very low ATPase activity (25,26,45), and it was suggested that transient dimer contacts near the amino terminus are required for tight binding of ATP and significant ATPase activity (45).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Heat Shock Protein 90 ATPase Activity by Sequences in the Carboxyl Terminus

Owen

Sullivan

Felts

et al. 2002

Journal of Biological Chemistry

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hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k cat of 0.02 min ؊1 and a K m greater than 300 M. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10 -100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k cat /K m ) was greater than 100 times that of the fulllength protein (k cat of 0.24 min ؊1 and K m of 60 M). In contrast, an internal deletion mutant, ⌬661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to fulllength hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.Heat shock protein 90 (hsp90) 1 has been demonstrated to be an important chaperone for a vast array of proteins involved in cell regulation, such as transcription factors and protein kinases (1-6). Interestingly, hsp90 seems to assist in the late stages of folding its substrate and may be important for modulating specific interactions such as ligand or co-factor binding or covalent modifications such as phosphorylation. hsp90 functions optimally in a multicomponent complex of chaperone proteins including hsp40, hsp70, Hop, p23, and one of a variety of immunophilins (1-6).Until recently, the evidence for nucleotide binding and hydrolysis by hsp90 was controversial. There is now direct evidence that hsp90 binds ADP and ATP and that the conformational state of hsp90 differs substantially depending upon which nucleotide is bound (7-11). The crystallization of the amino-terminal 220 amino acids of hsp90 with either nucleotide or an hsp90-specific inhibitor, geldanamycin, revealed that hsp90 possesses a unique nucleotide-binding site that differs substantially from that of hsp70 (8, 9). It has thus been proposed that hsp90 belongs to the GHKL superfamily of ATPbinding proteins that includes the DNA-repair protein MutL, DNA gyrase, topoisomerase II, and histidine kinases (12). The proposed mechanism of action for DNA gyrase and topoisomerase II requires transient ATP-mediated amino-terminal dimerization to form a "molecular clamp" that allows single-stranded DNA strand passage to occur. Binding of single-stranded DNA increases the ATPase activity of the enzyme by severalfold, and ATP hydrolysis is required to reset the "clamp" (13,14). It has been suggested that hsp90 may interact with ATP and its substrate (client protein) through a similar molecular clamp mechanism (4 -6).Although the binding site for adenine nucleotides has been localized t...

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“…When free in solution, hsp90 is dimerized through contacts near the C terminus, leaving the N-termini distant from one another (35). However, electron microscopy studies show a relatively linear or open hsp90 dimer in untreated samples that is converted to a circularized molecule by treatment with heat or ATP, suggesting new interactions near the N-termini (44). Also, an N-terminal fragment of yeast hsp90 has been crystallized as a dimer but with only a small region of dimer contacts that may or may not be of physiological importance (14).…”

Section: Discussionmentioning

confidence: 99%

Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90

Chadli

Bouhouche

Sullivan

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

122

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Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione-S-transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.H eat shock protein (hsp)90 is an abundant and ubiquitous molecular chaperone that is required to assist the conformational maturation of specific targets involved in many key functions of the cell such as cell-cycle regulation and signal transduction. Among hsp90 targets are several protein kinases such as v-Src, Weel, and c-Raf, transcriptional regulators such as p53 and steroid receptors, and two polymerases: that of hepatitis B virus and telomerase (for review, see refs. 1-4). To date, the best known maturation process driven by hsp90 is the assembly of steroid receptors into a high-affinity hormone-binding conformation. This complex, multistep process occurs in an ATPdependent manner and involves several other chaperones and co-chaperones (4). Three dynamic steps have been observed in this assembly process (5-7). The receptor initially associates with hsp70, assisted by hsp40. This association develops into an intermediate complex in which the co-chaperone, hsp organizing protein (Hop), associates with both hsp70 on the receptor and hsp90, while the protein Hip binds to hsp70. As the receptor complex matures, hsp90 bound to the receptor interacts with p23 and any one of four tetratricopeptide repeat-containing proteins, FK506-binding protein (FKBP)52, FKBP51, cyclophilin-40, or phosphoprotein phosphatase 5. The details of the interactions between these partner proteins are poorly understood and the mechanisms driving the transitions from one complex to another are still unresolved.hsp90 is a dimeric protein with multiple domains. Several proteins involved in the hsp90 chaperone pathway possess structurally related tetratricopeptide repeat motifs that mediated their interaction with hsp90 a...

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Monomer Arrangement in HSP90 Dimer as Determined by Decoration with N and C-Terminal Region Specific Antibodies

Cited by 71 publications

References 23 publications

Hsp90—From signal transduction to cell transformation

Hsp90—From signal transduction to cell transformation

Regulation of Heat Shock Protein 90 ATPase Activity by Sequences in the Carboxyl Terminus

Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90

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